Selection of linkers for a catalytic single-chain antibody using phage display technology

Phage display has been evaluated as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. Preliminary experiments with a conventional linker failed to yield a functional single-chain version of a catalytic antibody with chorismate mutase activity. A random linker library was therefore constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire ( approximately 5 x 10(6) different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers. There are apparently many viable solutions to the problem of linking individual VH and VL domains, but subtle differences in sequence dramatically influence the production, stability, and recognition properties of the scFv. The success of these experiments suggests that phage display will be generally useful for identifying peptide sequences for covalently linking any two protein domains.